J OURNAL OF C RUSTACEAN B IOLOGY, 36(2), 180-188, 2016
A ROSE BY ANY OTHER NAME: SYSTEMATICS AND DIVERSITY IN THE CHILEAN
GIANT BARNACLE AUSTROMEGABALANUS PSITTACUS
(MOLINA, 1782) (CIRRIPEDIA)
Paula Pappalardo 1,∗ , Fabio B. Pitombo 2 , Pilar A. Haye 3,4 , and John P. Wares 1,4
1 Odum
School of Ecology and Department of Genetics, University of Georgia, Athens, GA 30602, USA
de Biologia Marinha, Universidade Federal Fluminense, Niterói, RJ, Brazil, CEP 24020-141
3 Laboratorio de Diversidad Molecular, Departamento de Biología Marina, Facultad de Ciencias del Mar,
Universidad Católica del Norte and Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Coquimbo, Chile
4 Interdisciplinary Center for Aquaculture Research (INCAR), Universidad de Concepción, Concepción, Chile
2 Departamento
ABSTRACT
We analyzed the population structure of the edible barnacle Austromegabalanus psittacus (Molina, 1782) along most of the coast of Chile.
The analysis of population structure was based on nucleotide sequences of the mitochondrial cytochrome oxidase I (COI) gene region. We
also tested for differences between the regions to the north and south of 30-33°S, as these latitudes represent a recognized biogeographic
break and important oceanographic transitions occur in that area. No geographic differentiation was evident when using Hudson’s nearestneighbor (S nn ) statistic to analyze genetic differences between all populations. F st values nevertheless showed overall genetic structure
among sites. Significant geographic structure was found using S nn and analysis of molecular variance (AMOVA) when locations were
separated into northern and southern regions, with a stronger signal when the geographic division is set at 33°S. Our results support the
idea that oceanographic transitions can affect the genetic structure in species with pelagic larvae. We also discuss observations on size
structure differences within the natural range of A. psittacus and this barnacle’s sympatric occurrence with another barnacle, Megabalanus
concinnus (Darwin, 1854) in its northern range.
K EY W ORDS: genetic diversity, genetic structure, larval dispersal, phylogeography, picoroco
DOI: 10.1163/1937240X-00002403
I NTRODUCTION
Austromegabalanus psittacus (Molina, 1782), also locally
known as picoroco, is a commercially important barnacle,
exploited by local fisheries along the Chilean coast. It can
reach 20 cm in height (Pilsbry, 1916), and is one of the
few acorn barnacles worldwide that are eaten by humans.
Austromegabalanus psittacus is one of two species that have
undergone pilot farming by aquaculturists, with promising
results (López et al., 2010). Since the exploitation of A.
psittacus varies temporally and spatially (López et al.,
2012; SERNAPESCA, 2013) and natural populations of this
species appear to be periodically overharvested in Chile
(López et al., 2012), we were interested in evaluating the
genetic diversity and population structure of A. psittacus
along the Chilean coast.
Two main biogeographic provinces can be identified
along the Chilean coast (Fig. 1), the Peruvian Province to
the north of 30°S and the Magellanic Province south of 42°S,
with a transitional area from approximately 30 to 42°S degrees of latitude (Brattström and Johanssen, 1983; Camus,
2001). The exact location of the biogeographic break that
separates the Peruvian Province and the transitional area
varies depending on the taxonomic group analyzed: many
range limits for multiple taxonomic groups have been re∗ Corresponding
ported both at 30 and at 33°S (Brattström and Johanssen,
1983; Camus, 2001). Austromegabalanus psittacus spans
most of the Peruvian and Magellanic provinces (Fig. 1),
ranging along the South American coast from Lima, Peru
(12°S), along the entire Chilean Pacific coast, northwards
to 39°S on the Argentinian Atlantic coast (Pilsbry, 1909;
Young, 2000). It has also been reported on Juan Fernández
Island off Chile (Nilsson-Cantell, 1929). Hosie and Ahyong
(2008) reported the first observation of A. psittacus outside
its presently known natural range, a small clump of nine individuals that was found at Port Wellington, New Zealand,
its presence in New Zealand explained by ship transportation.
The broad linear distribution of this barnacle (about
7000 km) harbors much potential for natural variation in
form and genetic diversity. Pilsbry (1909) observed that
specimens of A. psittacus collected around Lima, Peru,
which he called the “Peruvian” form, were generally smaller
than southern Chilean specimens. Although we could not
find any further reference on natural variability of A.
psittacus along the coast, we did observe in the field that
A. psittacus individuals were smaller in northern Chile
(P. Haye, personal observation). If variation exists and is
heritable, this type of natural population variability could
affect future aquaculture efforts. For instance, the trial
author; e-mail: [email protected]
© The Crustacean Society, 2016. Published by Brill NV, Leiden
DOI:10.1163/1937240X-00002403
PAPPALARDO ET AL.: GENETIC DIVERSITY IN A GIANT BARNACLE
181
Fig. 1. Study area and locations sampled. Black dots indicate locations sampled for this study, white dots the locations of the museum voucher specimens
analyzed. The gray stars along the coast represent the distribution range of Austromegabalanus psittacus (Molina, 1782).
culture of this species at three sites along the Chilean
coast showed differences between the sites in the density
of juveniles collected and in growth rates (López et al.,
2012); these differences could have a genetic basis, or be
generated by the diverse environmental conditions along the
coast (Broitman et al., 2001; Hormazábal, 2004; Yuras et
al., 2005; Thiel et al., 2007). Given the spatial variation
in the exploitation of A. psittacus, it would be useful to
understand the scale of diversity within and between A.
psittacus populations along the coast prior to continued
exploration as a target for aquaculture.
The within-species genetic diversity of many taxa often
coincides with biogeographic breaks, because the genetic
structure of a species is a combination of historical and
present factors acting as barriers to dispersal that could also
contribute to speciation (Wares et al., 2001). Even sessile
marine species can exhibit high dispersal and low genetic
diversity, usually by means of a pelagic larval stage that
can last for months as in the edible snail Concholepas
concholepas (Bruguière, 1789) (Cárdenas et al., 2009). On
the other hand, species without a pelagic larval phase often
exhibit high population genetic structure (Sánchez et al.,
2011). Haye et al. (2014) compared the genetic structure
of a group of benthic marine invertebrate species with short
and long dispersal across the proposed biogeographic break
between the Peruvian Province and the intermediate area
(Fig. 1) and found diverse support for this pattern. Based
on the genetic structure found in the species with short
dispersal, Haye et al. (2014) proposed an ancient origin for
the 30°S break, while they found no genetic structure for the
species with long-lived pelagic larvae.
But despite the general increase in dispersal potential
with larval duration, a long larval duration also increases
the chances of being transported by currents to unfavorable
habitats (Marshall et al., 2010) or being lost offshore
(Gaylord and Gaines, 2000; Byers and Pringle, 2006).
Other studies that have analyzed phylogeographic breaks in
species with different larval duration suggest that while the
ranges of species with a limited capacity for dispersal could
reflect historical barriers to gene flow, species with long
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 36, NO. 2, 2016
larval duration tend to show phylogeographic breaks that
coincide with present-day oceanographic transitions (Pelc et
al., 2009; Altman et al., 2013).
Several oceanographic discontinuities have been described along the Chilean coast between 30 and 33°S that
can influence the phylogeographic structure of species with
a pelagic larva. The 30°S latitude separates two regions with
different wind stress and eddy kinetic energy (Hormazábal,
2004), and upwelling centers that can affect larval transport
(Shanks et al., 2000) have been described for sites at 31° and
33°S (Broitman et al., 2001; Aravena et al., 2014). Chlorophyll concentration is often used as a proxy for productivity
and could reflect food availability for pelagic feeding larva;
an alongshore change in chlorophyll concentration has been
reported at 33°S in coastal waters (Yuras et al., 2005). These
oceanographic changes coincide with changes in recruitment and abundance of marine invertebrates (Broitman et
al., 2001; Navarrete et al., 2005) and are reflected in the biogeography of coastal Chile (Brattström and Johanssen, 1983;
Fernández et al., 2000; Camus, 2001; Thiel et al., 2007) and
within-species genetic diversity (Haye et al., 2014).
Phylogeographic structure has been studied so far in two
barnacles along the Chilean coast, Jehlius cirratus (Darwin,
1854) and Notochthamalus scabrosus (Darwin, 1854). Contrasting patterns of genetic structure have been observed in
these two species (Zakas et al., 2009) despite their similar larval duration (Venegas et al., 2000). Notochthamalus
scabrosus exhibits strong latitudinal structure associated
with the biogeographic boundaries described, whereas J. cirratus has little population structure (Zakas et al., 2009). Because A. psittacus shows a typical balanid development with
six naupliar pelagic stages (López and Toledo, 1979), we
could expect that its larvae will spend weeks in the water.
For example, the larvae of its congener, Austromegabalanus
nigrescens (Lamarck, 1818), had a larval duration of 13-23
days at 20°C (Egan and Anderson, 1987). If A. psittacus has
a similar larval duration, it is likely that larval dispersal is
enough to maintain high connectivity and low genetic struc-
ture along the Chilean coast. But it is also likely that the
oceanographic discontinuities that have been shown to affect recruitment in barnacle larva at 32-33°S (Navarrete et
al., 2005) leave a signature in the genetic structure of A. psittacus.
We explore here the diversity of A. psittacus along the
Chilean coast from 18 to 42°S to identify any relevant population structure in this commercially important barnacle. In
particular we evaluated the genetic structure of A. psittacus
across the biogeographic and oceanographic transition reported between 30 and 33°S. We use our results to discuss
limits on how populations are interconnected regionally. Our
initial assessment of genetic structure could be important for
the management of this resource.
M ATERIALS AND M ETHODS
DNA Collection, Extraction, Amplification, and Sequencing
Large picoroco-like barnacles were obtained from field samples and
local fish markets between 18.5 and 42.5°S along the coast of Chile
(Fig. 1, black dots). Because not all of them appeared to belong to
Austromegabalanus psittacus, we sequenced specimens of A. psittacus
and Megabalanus concinnus (Darwin, 1854) deposited at the Museu
Nacional do Rio de Janeiro (MNRJ) from Chile and Peru (Fig. 1, localities
shown as white dots); additional information for each of the museum
samples and GenBank accession numbers are provided in Table S1 in the
Supplementary Material in the online edition of this journal, which can
be accessed via http://booksandjournals.brillonline.com/content/journals/
1937240x. Megabalanus concinnus belongs to the same subfamily as A.
psittacus and shares part of its geographic range, being common in the
low intertidal and subtidal in Peru and Chile (Pitombo, 2010). Detailed
information on the collection sites of this study, the museum samples
analyzed, and the number of samples from each location is presented in
Table 1.
Specimens of A. psittacus were stored in 95% ethanol until dissection.
We isolated DNA from somatic (cirral) tissue using a standard PureGene
(Gentra Systems) protocol and amplified the mitochondrial cytochrome
oxidase I (COI) gene region using taxon-optimized primers developed
for this study (PsittCOI-F 5 ATTTTTGGAGCCTGATCTGC; PsittCOIR 3 TCAAAATAGGTGTTGATATA). We developed specific primers to
increase the efficiency of the PCR reaction, but the universal primers of
Folmer et al. (2004) worked for initial amplification of A. psittacus and
M. concinnus DNA. PCR (polymerase chain reaction) amplification was
Table 1. Collection sites of Austromegabalanus psittacus (Molina, 1782) and Megabalanus concinnus (Darwin, 1854), the numbers of individuals
sequenced successfully for each location, and the species collected. Species identity was confirmed based on matches with museum voucher specimens
from the Museu Nacional do Rio de Janeiro (MNRJ).
Locality
Chala, Arequipa, Perú
Chala, Arequipa, Perú
Arenillas, Arica, Chile
Iquique, Chile
Iquique, Chile
Estrellita, Mejillones, Chile
Bandurrias, Taltal, Chile
Pan de Azúcar, Chile
La Herradura, Chile
Punta Chungo, Chile
San Antonio, Chile
Tome, Concepción, Chile
Lenca, Puerto Montt, Chile
Ancud, Chiloé, Chile
Isla Lilihuapi, Gulf of Ancud, Chile
Castro, Chiloé, Chile
Origin
MNRJ 25491-3
MNRJ 25489-90
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
MNRJ 25496
This study
MNRJ 25494-5
This study
Sampling
Latitude (S)
Longitude (W)
Field
Field
Field
Field
Field
Field
Field
Field
Field
Field
Fish market
Fish market
Field
Field
Field
Fish market
15°52
15°52
18°28
20°41
20°41
22°46
25°09
26°08
29°57
31°53
33°35
36°37
41°38
41°52
42°09
42°29
74°15
74°15
70°19
70°12
70°12
70°19
70°46
70°39
71°22
71°30
71°37
72°58
72°40
73°50
72°40
73°46
n
3
2
5
12
4
3
1
24
12
13
7
15
1
18
2
4
Species
A. psittacus
M. concinnus
A. psittacus
M. concinnus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
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PAPPALARDO ET AL.: GENETIC DIVERSITY IN A GIANT BARNACLE
performed following Wares et al. (2001) with an annealing temperature of
40°C. PCR products were purified using an ExoSAP reaction (BioLabs,
New England) and sequenced at Macrogen (http://www.macrogen.com).
DNA Analysis
Sequences were examined using CodonCode Aligner v.4.2.4 (http://www.
codoncode.com/aboutus.htm) and Geneious version 8.0.3 (http://www.
geneious.com; Kearse et al., 2012). The first quality cut was to include
only sequence reads in which at least half of the bases exhibited quality
scores larger than 40. The final sequence data were obtained by combining
forward and reverse reads of the same PCR amplicon (98.4% of cases),
including all A. psittacus sequences. In 1.6% of the cases, we used only one
read when the quality was good, as in the museum specimen MNRJ 25493
of A. psittacus and one sample of M. concinnus collected in this study from
Iquique, Chile. After combining the sequences, we regarded all nucleotides
that had quality scores lower than 20 as ambiguities, and we eliminated
sequences that were too short or had several ambiguities. We translated each
sequence to check that we had amplified the coding sequence of COI rather
than potential pseudogenes. A total of 126 sequences that passed the quality
tests were included in our analysis and are reported by location in Table 1.
GenBank accession numbers of all the sequences collected in this study are
provided in Table S2 in the Supplementary Material in the online edition of
this journal, which can be accessed via http://booksandjournals.brillonline.
com/content/journals/1937240x.
We assigned our samples to species using the museum samples as
reference, and tested the monophyly in phylogenetic groupings with
other barnacle species. After the first set of analyses we realized that
although most of the picoroco samples matched the museum voucher
of A. psittacus, the specimens collected from Iquique grouped with the
voucher specimen of M. concinnus. Consequently, we did not use the
latter samples for the analysis of genetic diversity of A. psittacus, but
we included them in a general comparison with other barnacle species.
To test the monophyly of the A. psittacus and M. concinnus clades we
analyzed our data along with GenBank reference sequences from species
of Balanus, Megabalanus, Amphibalanus, and Semibalanus; GenBank
accession numbers are provided in Supplementary Table S3 (Appendix).
Neighbor-joining analysis (bootstrap with 1000 replicates) was performed
in Geneious to identify clades supported in 95% or more of replicates.
Subsequently, monophyletic clades were evaluated using Rosenberg’s
test implemented in the species delimitation plugin of Geneious version
8.0.3 (http://www.geneious.com; see Kearse et al., 2012). There was no
ambiguity between samples of A. psittacus and M. concinnus, all samples
of each species conformed to monophyletic clades with 100% support.
Analysis of Geographic Structure
We performed the data analysis of geographic structure in R (R Core
Team, 2013), using the packages adegenet (Jombart, 2008), ape (Paradis
et al., 2004), muscle (Edgar, 2004), seqinr (Charif and Lobry, 2007), pegas
(Paradis, 2010), and PopGenome (Pfeifer et al., 2014). The corresponding
functions and packages for each analysis are described in the rest of this
section. Previous studies reported phylogeographic, biogeographic, and
oceanographic discontinuities in different locations between 30 and 33°S,
and we had one locality inside that zone (Punta Chungo, 31.8°S). For this
reason we ran the analyses of population structure twice, including Punta
Chungo in a “northern” group (separating samples at 33°S) or a “southern”
group (separating samples at 30°S).
The clade that matched the voucher specimen sequence data for A.
psittacus was further analyzed for population structure and genetic diversity
across the study area (Fig. 1). We analyzed only the sequences collected in
this study, without including the museum specimens or the single individual
from Taltal, Chile. We used the PopGenome package (Pfeifer et al., 2014)
to sort the sequences by geographic location and to estimate genetic
diversity (using the “F_ST.stats” methods) and Tajima’s D (using the
“neutrality.stats” methods). We tested the significance of Tajima’s D using
simulated populations created with the function “MS” in the PopGenome
package (Pfeifer et al., 2014).
To analyze genetic differences between A. psittacus populations we
estimated Hudson’s nearest-neighbor (S nn ) statistic, which evaluates how
often similar sequences belong to the same population (Hudson, 2000).
S nn was calculated across all sampling locations, and then by specifically
grouping sequences from individuals collected north and south of the 3033°S biogeographic break. To assess statistical significance, we randomized
the data matrix 1000 times and estimated the 95% confidence interval for
S nn based on the randomizations.
Table 2. Criteria used to separate populations of Austromegabalanus psittacus (Molina, 1782) in the analysis of geographic structure. Populations
were separated in “north” or “south” of 30-32°S to evaluate if there were
differences across this biogeographic break. Since Punta Chungo is in latitude 31.8°S, we ran the analysis twice, assigning Punta Chungo to the north
or to the south.
Group Locality
Latitude
(S)
Longitude
(W)
North
Arenillas, Arica, Chile
Iquique, Chile
Estrellita, Mejillones, Chile
Pan de Azúcar, Chile
La Herradura, Chile
18°28
20°41
22°46
26°08
29°57
70°19
70°12
70°19
70°39
71°22
5
4
3
24
12
Punta Chungo, Chile
31°53
71°30
13
San Antonio, Chile
Tome, Concepción, Chile
Ancud, Chiloé, Chile
Castro, Chiloé, Chile
33°35
36°37
42°09
42°29
71°37
72°58
72°40
73°46
7
15
18
4
South
n
Because we found significant geographic structure, we tested for
isolation by distance using a Mantel test implemented in the package
adegenet (Jombart, 2008) by the function “mantel.randtest” (using 5000
permutations to test for significance). A Mantel test compares the matrix of
genetic differences between pairs of locations with the matrix of geographic
distances between locations and tests for a linear relationship. The matrix
of genetic distance was created using the F st values computed in Arlequin
(Excoffier and Lischer, 2010); the matrix of geographic distance was
created in R using the function “rdist.earth” of the package fields (Nychka et
al., 2015). We additionally performed a hierarchical analysis of molecular
variance (AMOVA) to evaluate genetic variance among the regions to
the north and to the south of the 30-33°S biogeographic break (groups
defined in Table 2). We performed the AMOVA in Arlequin (Excoffier and
Lischer, 2010) using infinite site model-appropriate settings, and echoed
this analysis in R using the “amova” function in the pegas package (Paradis,
2010).
We constructed haplotype networks for the A. psittacus clade using the
minimum spanning network method implemented in PopArt (http://popart.
otago.ac.nz). We coded the haplotypes by sampling site and according to
region, north or south of the recognized biogeographic break at 30-33°S.
R ESULTS
We successfully sequenced a fragment of 651 base pairs (on
average) of the mitochondrial cytochrome oxidase I (COI)
region for 118 picoroco-like individuals collected from field
sites and local fish markets along the Chilean coast (Fig. 1,
Table 1). Of all the individuals sequenced, 106 individuals
unambiguously matched 6 sequences from museum voucher
specimens of Austromegabalanus psittacus. Twelve individuals collected in Iquique, however, were a clear match for
the voucher specimen sequences for Megabalanus concinnus. GenBank accession numbers for the museum specimen’s sequences of both species are provided in Table S1
in the Supplementary Material in the online edition of this
journal, which can be accessed via http://booksandjournals.
brillonline.com/content/journals/1937240x.
Because the phylogenetic signal for deeper phylogenetic
branches is weak for the COI gene region (Wares et al.,
2009), we did not evaluate the overall phylogeny (including
samples from this study together with related species from
GenBank), but we used it to investigate the monophyly of
the A. psittacus and M. concinnus clades. We found a wellsupported monophyletic clade for A. psittacus, and another
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 36, NO. 2, 2016
for M. concinnus (Rosenberg’s test for both clades, P <
0.0001).
The sequence data for A. psittacus ranged from 587 to
720 nucleotides in length. After alignment and editing, we
kept 614 sites for the analysis, exhibiting 65 variable positions. Across the 105 sequences of A. psittacus analyzed we
found a nucleotide diversity of 0.008 (π per nucleotide), a
Tajima’s D value of −1.58 (P = 0.02), and a haplotype
diversity of 0.97. The haplotype network showed no apparent geographic structure by collection site in the A. psittacus
clade (Fig. 2).
The analysis of geographic structure included all samples of A. psittacus collected during the investigation (Table 2) with the exception of the sample from Taltal, which
was excluded given the single available sample. The Hudson’s nearest-neighbor (S nn ) statistic, estimated considering all the populations sampled (S nn = 0.13), was not significantly different from the value of S nn expected by a
95% probability distribution of randomized values (5% =
0.09, 95% = 0.18, P = 0.488). We nevertheless observed
overall genetic structure in the F st values, full AMOVA
table provided in Table S4 in the Supplementary Material in the online edition of this journal, which can be ac-
cessed via http://booksandjournals.brillonline.com/content/
journals/1937240x.
When sequences were separated into northern and southern regions by placing the geographic division at 30°S, the
genetic diversity of each region was similar (0.0073 in the
north and 0.0084 in the south), but statistically significant
geographic structure was observed between the two regions.
The observed S nn was 0.59, with the 95% distribution of
randomized values from 0.43 to 0.56 (P = 0.012). The
other regional partition for the proposed location of the biogeographic transition at 33°S generated similar results. The
largest value of S nn was found when Punta Chungo (31.8°S)
was included in the northern group, and the division set at
33°S.
When the geographic division representing the biogeographic break is represented in a haplotype network (Fig. 3,
showing samples from north or south of the 33°S break),
there are no clear genetic groups associated with a particular region, other than a small clade of “southern” haplotypes
(including samples from Concepción, San Antonio, and Ancud; this pattern is unchanged whether the division between
“north” and “south” is at 30 or 33°). When the break is set
at 33°S and Punta Chungo is considered in the northern region, we can also see a small clump of only northern hap-
Fig. 2. Haplotype network of COI haplotypes for Austromegabalanus psittacus (Molina, 1782). Circle size is proportional to the frequency of each
haplotype in the population. Sampling locations along the coast are represented by different colors. The haplotype network was constructed using PopArt
(http://popart.otago.ac.nz).
PAPPALARDO ET AL.: GENETIC DIVERSITY IN A GIANT BARNACLE
185
Fig. 3. Haplotype network of COI haplotypes for Austromegabalanus psittacus (Molina, 1782). Circle size is proportional to the frequency of each
haplotype in the population. Sampling locations along the coast were separated in two groups, north (light gray) or south (dark gray) of 32°S. The dashes
represent the number of mutational steps between haplotypes.
lotypes that includes samples from Iquique, Pan de Azúcar,
and Punta Chungo (Fig. 3).
The results of the AMOVA comparing the northern
and southern regions varied depending on the choice of
the geographic partition, the full AMOVA table is presented in Table S4 in the Supplementary Material in the
online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/
journals/1937240x. When the geographic partition was set
at 30° there were no significant differences between the
northern and southern groups (P 30°S = 0.076). When the
geographic partition was instead set at 33°S (with Punta
Chungo included in the “northern” group), there was a significant difference between the northern and southern regions (P 33°S = 0.011). These results corroborate the regional
geographic differentiation found with the S nn index. Our results considering all pairwise comparisons do not, however,
support a pattern of isolation by distance (P = 0.132).
D ISCUSSION
The modest phylogeographic structure of Austromegabalanus psittacus found along most of the Chilean coast suggests a generally high dispersal potential of this species
and high realized connectivity across populations. While
the haplotype network and the overall analysis suggest only
slight variation in the COI region of the mitochondrial
genome of A. psittacus populations, the analysis by region
marginally supports a differentiation of a northern and southern region, with a stronger signal when the division is set
at 33°S. The concordance of this signal with biogeographic
and oceanographic transitions along the Chilean coast is
compelling, since the geographic structure of A. psittacus
observed in part of our analysis coincides not only with
the biogeographic break around 30-33°S but also with the
abrupt changes in recruitment of barnacles reported at 3233°S (Navarrete et al., 2005).
Previous phylogeographic studies comparing genetic
structure across the 30°S break show different support for
a genetic break associated to the dispersal potential of the
species analyzed (Cárdenas et al., 2009; Zakas et al., 2009;
Sánchez et al., 2011; Brante et al., 2012; Laughlin et al.,
2012; Haye et al., 2014). Haye et al. (2014) compared the genetic structure of marine invertebrates having differing dispersal potential distributed across the 30°S biogeographic
break and showed that there is a coincident genetic break
only on species with low dispersal potential, suggesting that
gene flow of high dispersers has erased the signatures of the
break, which is strong evidence of the break having a historic origin. The exact location of the break in Haye et al.
(2014), however, varied between groups, with the asteroid
Stichaster striatus Müller & Troschel, 1840 and the gastropod Tegula atra (Lesson, 1830) showing a genetic break at
approximately 32°S. The reported larval duration was five
weeks for Stichaster striatus and one week for Tegula atra
(Haye et al., 2014), which suggests that the larvae of these
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 36, NO. 2, 2016
species could be affected by the oceanographic transitions in
this particular area.
Our observations of modest genetic structure between the
northern and southern populations of A. psittacus place this
species in the middle with respect to the other barnacles
studied on the Chilean coast. Jehlius cirratus shows only
slight genetic structure along the coast (Zakas et al., 2009;
J. P. Wares, personal observation), whereas Notochthamalus
scabrosus shows an important evolutionary shift in mitochondrial diversity at around 31°S (Zakas et al., 2009).
These contrasting results, even between related species, have
been observed with other barnacles on the Californian coast
(Sotka et al., 2004; Wares and Castañeda, 2005). At this
point it is difficult to explain why some barnacles, most of
which have high potential for larval dispersal, exhibit population structure (Sotka et al., 2004; Zakas et al., 2009; Govindarajan et al., 2015) and others do not (Wares and Castañeda,
2005), but some studies point to the important effect of larval
behavior on larval transport and recruitment (Marko, 2004;
Marshall et al., 2010) and it is quite likely that environmental
selection is involved as well (Sotka et al., 2004; Pringle and
Wares, 2007; C. Ewers-Saucedo et al., unpublished work).
Based on the lack of structure in the haplotype network
by site (Fig. 2) and the high nucleotide diversity, it appears
that A. psittacus has no problem dispersing its larvae across
broad distances to maintain connectivity among populations.
The nucleotide diversity of 0.8% found in this study falls
within the range reported for other barnacles (Ewers and
Wares, 2012), and shows no significant difference between
the regions to the north or to the south of the 30-33°S
break. Likewise, the estimate of Tajima’s D statistics for A.
psittacus is −1.58 (P = 0.02), also comparable to values
reported in other barnacle species (Ewers and Wares, 2012),
which tend to have a consistent pattern in deviation from
neutrality in the COI marker (Ewers and Wares, 2012).
Our results from the COI mitochondrial region suggest high
gene flow between populations, as could be expected if A.
psittacus exhibits a larval development similar to that of its
congener A. nigrescens (cf., Egan and Anderson, 1987). If
the high dispersal potential for A. psittacus is confirmed,
the management efforts of this edible species could focus on
the whole coast rather than on particular populations. Given
that we found slight geographic structure in the analysis
by regions, and that in our study only one marker was
analyzed, we recognize that further analysis is warranted
if the management of this resource requires movement of
stocks.
The spatial genetic structure observed in this study could
also justify further investigation into the observation of Pilsbry (1909, 1916) of a smaller variety of A. psittacus occurring on the Peruvian coast. Our observations of smaller individuals in some of the northern localities give some support to Pilsbry’s remark. Our sampling unfortunately did not
include Peru, which could allow more direct assessment of
Pilsbry’s observations on contemporary populations. Information on the presence of A. psittacus along the Peruvian
coast is scarce, mostly related to studies of community composition on rocky shores (Paredes, 1974; Paredes and Tarazona, 1980). We think that the variation in size of A. psittacus should be further explored, and that it could be related
to sympatry with M. concinnus in northern Chile and on the
Peruvian coast, since both species are likely to share similar life history traits and could exhibit displacement in size
structure. Both species were found at the same spot on the
pier pilings in Chala, Peru, where M. conccinus overall was
larger in size than A. psittacus (F. B. Pitombo, personal observation).
Sampling in Iquique showed that not all picorocos belong
to the same species. The samples were collected by a local diver, who was convinced he was sampling the Chilean
picoroco. Both Austromegabalanus and Megabalanus are
included in the subfamily Megabalaninae Newman, 1979,
which is characterized by the presence of wide tubiferous
radii recognizable only on disarticulated shells. The main
features that distinguish both genera are the position of denticles on the sutural edges of the radii, Megabalanus with
denticles on both sides and Austromegabalanus mostly on
the lower side only (Newman, 1979). An external characteristic, the pronounced tergal beak, is an easy character to
identify on A. psittacus (Fig. 4B, C, D); it is absent on M.
concinnus (Fig. 4H, I). The shell also shows other distinct
features, being more cylindrical in A. psittacus than in M.
concinnus, having a hexagonal aperture, and a light pink or
flesh color. In contrast, the shell is globular-conical with a
rounded aperture and a distinct freckled pattern of white and
purple in M. concinnus (Fig. 4A, B, G) (Pitombo, 2010).
Other features can be used to separate the two species, such
as the spur position, which is close to the basi-scutal angle
in A. psittacus but separate in M. concinnus (Fig. 4C, D, H,
I), and although both species present a prominent adductor
ridge in the scutum, it is confluent with an articular ridge
in A. psittacus, but separate in M. concinnus (Fig. 4F, K).
Despite these distinctive characteristics and given that there
is some overlap in size range, the two species could resemble each other during casual visual examination, especially
if they are covered with sediment or algae.
Our record of M. concinnus in Iquique, together with
the report of Pitombo (2010) from Arica, demonstrates the
presence of this species in northern Chile. As Pitombo
(2010) pointed out, the presence of M. concinnus along the
rest of the Chilean coast needs to be investigated. There has
been only one such record until now, that of Gruvel (1903) in
the Strait of Magellan, where M. concinnus specimens were
found on A. psittacus. Since all the aquaculture efforts and
pilot studies have been centered on A. psittacus (López et al.,
2012), even though M. concinnus is present in northern Chile
(and probably is being harvested there), it could be useful in
order to gain additional knowledge on how the two species
interact in their overlapping range.
This study contributes new COI sequence data for two
barnacle species (A. psittacus and M. concinnus) that have
not been represented until now in GenBank and could,
therefore, be of use in future phylogenetic and taxonomic
studies of this group. The genetic structure detected for A.
psittacus between the regions to the north and south of
the 30-33°S break could be further explored with a more
diverse collection of individuals and loci, sampling in more
sites within and outside the 30-33°S area, in order to better
understand the factors associated with regional biodiversity
transition.
PAPPALARDO ET AL.: GENETIC DIVERSITY IN A GIANT BARNACLE
187
Fig. 4. Shell and opercular plates of Austromegabalanus psittacus (Molina, 1782) (A-F) and Megabalanus concinnus (Darwin, 1854) (G-K). A, B, and G,
shell view; C, H, and D, I, left terga outer and inner view, respectively; E, J, and F, K, left scuta outer and inner view; bk, tergal beak; ar, articular ridge; adr,
adductor ridge; bk, tergal beak; bsa, basi-scutal angle; spr, tergal spur. Scale bars: A, B, G = 2 cm; C-F and H-K = 1 cm.
ACKNOWLEDGEMENTS
The authors would like to thank Stella Januario, Kennia Morales, Bryan
Bularz, Mirtala Parrague, Mayra Figueroa, Lucy Travo, Onofre Correa,
and Natalia Muñoz, who helped with the collection of tissue samples
and specimens, and Hayley Glassic and Alice Vislova, who helped with
part of the sequencing process. We thank our funding sources, NSFOCE-1029526 to JPW, Fondecyt grants 1090670, 114682, and INCAR
(FONDAP 15110027) to PAH, and a CAPES (BEX 5841/14-3) grant
to FBP. We received extensive technical help from Katie Bockrath and
Christine Ewers-Saucedo at different stages of the preparation of this
manuscript. We thank three anonymous reviewers and the associate and
general editors for their suggestions that greatly improved the quality of
the manuscript. Finally, thanks to R and all the package contributors that
make free and reproducible science possible.
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R ECEIVED: 16 June 2015.
ACCEPTED: 10 December 2015.
AVAILABLE ONLINE: 28 January 2016.
S1
2 m depth, attached to pier pilings
2 m depth, attached to pier pilings
2 m depth, attached to pier pilings
2 m depth, attached to pier pilings
2 m depth, attached to pier pilings
15°51 54 S, 74°14 58 W
15°51 54 S, 74°14 58 W
15°51 54 S, 74°14 58 W
15°51 54 S, 74°14 58 W
15°51 54 S, 74°14 58 W
42°09 30 S, 72°36 14 W
42°09 30 S, 72°36 14 W
41°38 01 S, 72°40 07 W
Chala, Arequipa, Peru
Chala, Arequipa, Peru
Chala, Arequipa, Peru
Chala, Arequipa, Peru
Chala, Arequipa, Peru
Isla Liliahupi, Golfo de Ancud, Chile
Isla Liliahupi, Golfo de Ancud, Chile
Punta Chaicas, Lenca, Puerto Montt, Chile
13 October 1999
13 October 1999
13 October 1999
13 October 1999
13 October 1999
25 March 2003
25 March 2003
5 March 2007
M. concinnus
M. concinnus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
25489
25490
25491
25492
25493
25494
25495
25496
Coordinates
Locality
Collection date
Species
9 m depth
GenBank
accession number
KU160497
KJ769122
KJ756013
KJ756014
KU144718
KJ756083
KJ756065
KJ756010
MNRJ
Observations
Voucher specimens of Austromegabalanus psittacus (Molina, 1782) and Megabalanus concinnus (Darwin, 1854) deposited in the Museu Nacional do Rio de Janeiro (MNRJ).
Table S2. GenBank accession numbers, corresponding label, and collecting sites of all the samples of Austromegabalanus psittacus (Molina, 1782)
and Megabalanus concinnus (Darwin, 1854) sequenced.
Table S1.
Supplementary Material
PAPPALARDO ET AL.: GENETIC DIVERSITY IN A GIANT BARNACLE
GenBank
accession
number
Label
Site
Species
KJ756058
KJ756036
KJ756037
KJ756088
KJ756006
KJ756081
KJ756052
KJ756032
KJ756047
KJ756091
KJ756003
KJ756022
KJ756004
KJ756093
KJ756060
KJ756059
KJ756074
KJ756048
KJ756044
KJ756023
KJ756076
KJ756017
KJ756054
KJ756046
KU144736
KU144739
KU144738
KU144737
KJ756027
KJ756049
KJ756018
KJ756030
KJ756073
KJ756033
KJ756078
KJ756057
KU144716
KJ756021
KU144715
KJ756040
KJ756024
KU144717
KJ756090
KJ756002
KJ756067
KJ756035
KJ756009
KJ756055
KJ756071
KJ756028
KJ756043
KJ756092
KJ756095
KJ756038
KJ756034
KJ756039
KJ756077
KJ756019
Anc_1
Anc_10
Anc_11
Anc_12
Anc_13
Anc_14
Anc_15
Anc_18
Anc_19
Anc_2
Anc_20
Anc_21
Anc_3
Anc_4
Anc_5
Anc_7
Anc_8
Anc_9
Ari_1
Ari_2
Ari_3
Ari_4
Ari_5
Tal_1
Cas_10
Cas_6
Cas_7
Cas_8
Mej_1
Mej_3
Mej_4
Iqu_17
Iqu_18
Iqu_19
Iqu_20
Her_1
Her_10
Her_11
Her_14
Her_2
Her_3
Her_4
Her_5
Her_6
Her_7
Her_8
Her_9
Pan_11
Pan_12
Pan_13
Pan_14
Pan_15
Pan_16
Pan_17
Pan_18
Pan_19
Pan_2
Pan_20
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Ancud, Chiloé, Chile
Arenillas, Arica, Chile
Arenillas, Arica, Chile
Arenillas, Arica, Chile
Arenillas, Arica, Chile
Arenillas, Arica, Chile
Bandurrias, Taltal, Chile
Castro, Chiloé, Chile
Castro, Chiloé, Chile
Castro, Chiloé, Chile
Castro, Chiloé, Chile
Estrellita, Mejillones, Chile
Estrellita, Mejillones, Chile
Estrellita, Mejillones, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
La Herradura, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
S2
Table S2.
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 36, NO. 2, 2016
(Continued.)
GenBank
accession
number
Label
Site
Species
KJ756045
KJ756087
KJ756031
KJ756050
KJ756094
KJ756079
KJ756056
KJ756005
KJ756069
KJ756086
KJ756075
KJ756085
KJ756015
KJ756007
KU144734
KJ756041
KJ756089
KJ756051
KJ756011
KJ756063
KJ756042
KJ756082
KU144735
KJ756012
KJ756080
KJ756072
KU144711
KU144705
KU144710
KU144709
KU144708
KU144707
KU144706
KU144733
KU144725
KU144724
KU144723
KU144722
KU144721
KU144720
KU144719
KU144732
KU144731
KU144730
KU144729
KU144728
KU144727
KU144726
KU144714
KJ769113
KJ769114
KJ769115
KJ769112
KJ769120
KU144713
KJ769121
KU144712
KJ769116
KJ769119
KJ769117
Pan_22
Pan_23
Pan_24
Pan_25
Pan_26
Pan_27
Pan_3
Pan_32
Pan_33
Pan_34
Pan_5
Pan_6
Pan_9
Chu_10
Chu_12
Chu_14
Chu_15
Chu_16
Chu_17
Chu_18
Chu_19
Chu_20
Chu_3
Chu_5
Chu_6
Chu_7
San_1
San_10
San_2
San_4
San_5
San_7
San_8
Con_1
Con_10
Con_11
Con_12
Con_13
Con_14
Con_15
Con_16
Con_2
Con_3
Con_4
Con_6
Con_7
Con_8
Con_9
Iqu_1
Iqu_10
Iqu_11
Iqu_13
Iqu_14
Iqu_16
Iqu_2_F
Iqu_3
Iqu_5
Iqu_6
Iqu_7
Iqu_8
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Pan de Azúcar, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
Punta Chungo, Chile
San Antonio, Chile
San Antonio, Chile
San Antonio, Chile
San Antonio, Chile
San Antonio, Chile
San Antonio, Chile
San Antonio, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Tome, Concepción, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
Iquique, Chile
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
A. psittacus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
M. concinnus
Table S3. GenBank accession numbers and species names of the acorn
barnacles whose COI sequences were compared with those of the Austromegabalanus psittacus (Molina, 1782) clade. ∗ Perforatus perforatus is
registered as Balanus perforatus in GenBank.
GenBank accession number
Species
JQ035515
JQ035516
KC138445
JQ035520
JQ035522
KC138447
KC138448
HM029124
HM029125
HM029126
KF297561
KF297562
KF297563
JQ035524
KC138451
KC138452
KC138491
KC138479
KC138480
HG970519
KC138481
KC138482
KC138483
KC138484
JX503004
JX503005
KC138485
KC138486
JQ035527
KC138487
KC138488
KC138489
KC138490
KC138492
FJ845815
FJ845819
GU442631
GU442641
GU442643
KC935856
KC935857
DQ363697
DQ363699
Amphibalanus amphitrite
Amphibalanus amphitrite
Amphibalanus amphitrite
Amphibalanus variegatus
Amphibalanus variegatus
Amphibalanus variegatus
Amphibalanus zhujiangensis
Balanus glandula
Balanus glandula
Balanus glandula
Perforatus perforatus∗
Perforatus perforatus∗
Perforatus perforatus∗
Balanus trigonus
Balanus trigonus
Balanus trigonus
Mebalanus zebra
Megabalanus ajax
Megabalanus ajax
Megabalanus coccopoma
Megabalanus coccopoma
Megabalanus coccopoma
Megabalanus occator
Megabalanus occator
Megabalanus rosa
Megabalanus rosa
Megabalanus rosa
Megabalanus rosa
Megabalanus tintinnabulum
Megabalanus tintinnabulum
Megabalanus tintinnabulum
Megabalanus volcano
Megabalanus volcano
Megabalanus zebra
Semibalanus balanoides
Semibalanus balanoides
Semibalanus cariosus
Semibalanus cariosus
Semibalanus cariosus
Tetraclita serrata
Tetraclita serrata
Tetraclita squamosa
Tetraclita squamosa
S3
PAPPALARDO ET AL.: GENETIC DIVERSITY IN A GIANT BARNACLE
Table S4. Results of the analysis of molecular variance (AMOVA) for the COI mitochondrial region comparing the regions to the north or to the south of
the 30-33°S break in the Chilean coast. ∗ P < 0.05.
Geographic
division
Source of variation
df
Sum of
squares
Variance
components
Percentage
of variation
Fixation
indices
P -value
30°S
Among groups
Among populations within groups
Within populations
Total
1
8
95
104
8.846
28.868
271.353
309.067
0.09193
0.07999
2.85635
3.02827
3.04
2.64
94.32
0.03036
0.02724
0.05677
0.07625
0.08798
0.00782∗
33°S
Among groups
Among populations within groups
Within populations
Total
1
8
95
104
12.985
24.728
271.353
309.067
0.1910
0.02482
2.85635
3.07223
6.22
0.81
92.97
0.06219
0.00861
0.07027
0.01075∗
0.28055
0.01075∗